Most conventional methods for detecting unexpected sequence variations require gel electrophoresis after PCR. These include single-strand conformation polymorphism (Orita, O., et al., Proc. Natl. Acad. Sci. USA. 86:2766-2770, 1989), heteroduplex migration analysis (Nataraj, A. J., et al., Electrophoresis. 20:1177-1185, 1999), denaturing gradient gel electrophoresis (Abrams, E. S., et al., Genomics 7:463-475, 1990), temperature gradient gel electrophoresis (Wartell, R. M., et al., J. Chromatogr. A. 806:169-185, 1998), and enzyme cleavage methods (Hawkins G. A., et al., Electrophoresis, 20:1171-1176, 1999). Identifying new mutations by DNA sequencing also requires multiple steps, including cycle sequencing and gel electrophoresis. Denaturing high-performance liquid chromatography (Narayanaswami, G., et al., Genetic Testing. 5:9-16, 2001) is a more recent method, but requires sampling and injection after PCR.
Recently, homogeneous fluorescent methods have been reported for mutation scanning. SYBR Green I is a double stranded DNA binding dye that is often used to monitor product formation (Wittwer C. T., et al., BioTechniques, 22:130-138, 1997) and melting temperature (Ririe K. M., et al., Anal. Biochem, 245:154-160, 1997) in real-time PCR. Following PCR product purification and addition of SYBR Green I, single nucleotide polymorphisms have been detected in up to 167 bp products by melting curve profiles (Lipsky, R. H., et al., Clin. Chem. 47:635-644, 2001). However, the high concentration of SYBR Green I used inhibits PCR (Wittwer C. T., et al., Methods, 25:430-442, 2001), so the dye was added after amplification. In addition, PCR product purification was necessary, further limiting the possibility of real-time analysis. In another report, GC clamping was used with SYBR Green I to detect single nucleotide polymorphisms in up to 212 bp products (Elenitoba-Johnson, K. S. J., et al., Am. J. Pathol. 159:845-853, 2001, and U.S. Pat. No. 6,346,386). However, after PCR, the solution required adjustment to 12M in urea before the melting analysis. In both cases, sample additions after PCR were necessary. Any manipulation of the sample increases the risk of PCR product carryover into subsequent reactions.
Another homogeneous fluorescent approach is to use real-time hybridization probes (Wittwer C. T., et al., BioTechniques, 22:130-138, 1997). These probes can detect any mutation under the probe by melting temperature shifts. Multiple single-labeled hybridization probes have been tiled across amplicons to scan for p53 mutations by Tm multiplexing (Millward H., et al., Clin. Chem, in press, 2002).
There are a number of designs for PCR primers that facilitate change in fluorescence when the primers are incorporated into the PCR product. These designs include but are not limited to the double-stranded displacement primer (Li, Q., et al., Nucleic Acids Res., 30: e5, 2002) whose fluorescence is quenched initially by an acceptor fluorophore placed on the complementary oligonucleotide that dissociates upon PCR, releasing the fluorescence signal, and the Scorpion primer (Whitecombe, D., et al., Nature Biotechnology, 17:804-807, 1999) which has a stem-loop tail that brings the reporter close to a quencher prior to PCR, but releases the signal by denaturation and incorporation into the PCR product. Most of these designs aim primarily to detect amplification. In some cases, genotyping had been performed by allele-specific amplification. None of these references teach the use of melting analysis and differentiation of sequence variation by melting temperature.
PCR primers fluorescently labeled at the 5′ residue have already been discussed to distinguish between different analytes based on differences in melting temperature (U.S. patent application 20010000175 Kurane et al). However, according to the teaching of Kurane, it is impossible to discriminate between small differences of sequence variants, since the results of a melting curve analysis very strongly depend on the concentration of the target nucleic acid. Therefore, prior to the present invention, it has never been shown that these, or other forms of labeled primers, can be used to detect small sequence variations or heteroduplexes in amplified product by melting analysis.
In one aspect, the present invention is directed to a simple and sensitive real time PCR method for mutation scanning and identification of small sequence variations on a broad range of sequences. By using a 5′-labeled PCR primer, single-nucleotide polymorphisms and other small sequence variances in PCR products can be detected by the melting profiles of the amplified product. These melting profiles show when a heteroduplex is present, and the melting profiles can be used for real time mutation scanning without any need for additions or manipulations after PCR. In addition, different homozygotes can often be distinguished from each other, as well as different heterozygotes. That is, genotyping is often possible with the methods of the present invention. Finally, subtyping of organisms as well as genetic haplotyping are possible based on the inventive method.
In one embodiment, the invention provides a method for sequence variation scanning that requires only PCR and amplicon melting analysis without any intermediate processing. At least one of the PCR primers is fluorescently-labeled such that a change in fluorescence occurs when the amplicon is melted. Heteroduplexes are detected as a low-temperature shoulder and broadening of the peak on derivative melting curve plots. Heteroduplex detection is increased by denaturation, followed by rapid cooling (>2° C./s) before melting, low cation concentration, and rapid heating during melting (0.1-0.5° C./s). In an alternative embodiment, two unlabeled primers can be used with a third labeled indicator primer that has sequences homologous to a universal tail added to one of the unlabeled primers.
In various embodiments, the methods of the present invention have been used to detect polymorphisms in HTR2A (T102C), beta-globin (Hb S, C and E), apo E (2/2, 2/3, 2/4, 3/3, 3/4, and 4/4), and CFTR (F508del, F508C, I507del, I506V). In most cases, different homozygotes could be distinguished from each other by melting temperature (Tm), heterozygotes could be distinguished from homozygotes by a low temperature shoulder and a more gradual transition, and different heterozygotes could be differentiated from other heterozygotes by the shape of the fluorescent melting curve. Amplicon sizes varied from 44-303 bp. The presence of less than 5% variant DNA (differing at a single base in a 243 bp amplicon) was detected.